Paneth cells (PCs), which reside intercalated among the active crypt base columnar (CBC) intestinal stem cells (ISCs), serve several important homeostatic functions in the gut, including i) regulation of the intestinal microbiome by secretion of antimicrobial peptides and ii) serving as niche cells to provide growth factors and signals to adjacent CBC cells. Recent evidence has suggested that secretory cells (fated to become PCs) can revert to a CBC state to repopulate the intestine following injury. CBC cells are highly dependent on canonical Wnt signaling to drive proliferation and self-renewal. PCs likewise exist in a high-Wnt environment, and show robust nuclear ?-catenin indicative of canonical Wnt activation--but they do not proliferate. This raises the question: how does potent canonical Wnt activation produce such different results in adjacent epithelial cells? Our previously published results identified a transcriptional cascade (Atoh1-Gfi1-SPDEF) that specifies intestinal secretory cells including PCs. Our recent publications indicate that SPDEF (SAM Pointed Domain ETS Factor), which is normally expressed in PCs but not CBCs, can repress ?-catenin chromatin binding and transcriptional activation of select target genes. These results support our overarching hypothesis that SPDEF shifts the chromatin targets of ?-catenin to regulate stem cell vs. Paneth cell fate. We will test this hypothesis in three aims. Aim 1: Define the landscape of ?-catenin and SPDEF targets in CBCs vs. Paneth cells. We will use RNA- and Chromatin Immunoprecipitation- (ChIP-) seq to define functional targets of ?-catenin and SPDEF in Lgr5+ CBC stem cells and PCs. This aim will give us the first compendium of cell-specific targets of SPDEF and ?-catenin in the intestine, and test whether SPDEF is necessary and sufficient to shift the chromatin targets of ?-catenin in the stem cell niche. Aim 2: Determine the mechanism by which SPDEF changes transcription of ?-catenin targets. We will probe the physical interaction between SPDEF and ?-catenin protein complexes, as well as the changes to transcriptional machinery regulating CBC vs. PC genes. This aim will rigorously test our mechanistic hypothesis about how SPDEF alters ?-catenin targets, and is essential for any rational targeting of this process for therapeutic intervention. Aim 3: Test the role for SPDEF:?-catenin interaction in regeneration of damaged crypts. Here, we will use single-cell RNA-seq (scRNA-seq) and ChIP to characterize injury-induced reversion, and to determine how ?-catenin chromatin binding changes as part of the reversion process. Next, we will define the requirement and sufficiency for downregulation of SPDEF to achieve reversion of secretory cells into stem cells following injury. Finally, we will use a novel in vitro injury-reversion-regeneration methodology in organoids to test the role for SPDEF in regulating ?-catenin directed CBC recovery. This aim will provide single-cell resolution to define the mechanism of cellular plasticity within the crypt and the role of SPDEF and ?-catenin/WNT signaling in regeneration.